This project is directed at the role of the connecting cilium in disc assembly in photoreceptor. Disc assembly involves synthesis of phospholipid and membrane precursors in the photoreceptor inner segment, delivery of those components to the periciliary region, trans-ciliary transport of precursors and assembly of new discs. Understanding the molecular and cellular details f these processes is an important problem in photoreceptor physiology. A growing body of evidence suggest that derangement of disc assembly can lead to photoreceptor degeneration and impaired vision. Although much has been learned about synthesis and transport of precursors to the periciliary region and the formation of new discs by evagination of the distal ciliary membrane, the mechanism of trans-ciliary transport and the role of the connecting cilium in disc assembly remains undefined. A principal thesis of this proposal is that understanding the role of the cilium in disc assembly will require more detailed information on the structure and molecular composition of the connecting cilium itself. We propose to obtain this information by focusing on the organization of Y-shaped cross-linkers that link ciliary membrane components to doublet microtubules of the photoreceptor axoneme. We will take advantage of recently acquired information indicating that native cell surface glycoconjugates of the cilium are linked to doublet microtubules via the Y-shaped cross-linkers. Using the ligand binding properties of native glycoconjugates, we will attempt to isolate the cross-linkers, to define their polypeptide composition, and to raise monoclonal antibodies as structural probes. A second thesis is that to understand the role of cilium in disc assembly, we must conduct physiological experiments that test alternative hypotheses for trans- ciliary transport of disc membrane precursors. We will use isolated Xenopus and rat retinas in culture under conditions known to support new disc assembly and will use electron microscopic and biochemical techniques to study trans-ciliary transport. In these experiments we will consider the problems of phospholipid and protein transport independently because of evidence suggesting that the two principal components of discs are not oligately co-transported. This approach will add to our understanding of the fundamental mechanisms of disc assembly and may contribute to the more general problem of distal ciliary membrane assembly in other ciliated cells.